An individual needs to have a dynamic immune system that is able to adapt rapidly and respond adequately to potentially harmful microorganisms, and to respond to the exposure of a highly diverse and continuously changing environment. Higher organisms have evolved specialized molecular mechanisms to ensure the implementation of clonally-distributed, highly diverse repertoires of antigen-receptor molecules expressed by cells of the immune system: immunoglobulin (Ig) molecules on B lymphocytes and T cell receptors on T lymphocytes. A primary repertoire of (generally low affinity) Ig receptors is established during B cell differentiation in the bone marrow as a result of rearrangement of germ line-encoded gene segments. Further refinement of Ig receptor specificity and affinity occurs in peripheral lymphoid organs where antigen-stimulated B lymphocytes activate a somatic hypermutation machinery that specifically targets the immunoglobulin variable (V) regions. During this process, B cell clones with mutant Ig receptors of higher affinity for the inciting antigen are stimulated into clonal proliferation and maturation into antibody-secreting plasma cells (reviewed in Berek and Milstein. 1987).
Recombinant DNA technology has been used to mimic many aspects of the processes that govern the generation and selection of natural human antibody repertoires (reviewed in Winter and Milstein. 1991; Vaughan et al. 1998). The construction of large repertoires of antibody fragments (such as Fab fragments or single chain Fv fragments, scFv's) expressed on the surface of filamentous phage particles and the selection of such phages by “panning” on antigens has been developed as a versatile and rapid method to obtain antibodies of desired specificities (reviewed in Burton and Barbas. 1994). A subsequent optimization of the affinity of individual phage antibodies was achieved by creating mutant antibody repertoires of the selected phages and sampled for higher affinity descendents by selection for binding to antigen under more stringent conditions (reviewed in Hoogenboom. 1994).
M13 and M13-derived phages (sometimes also called viruses) are filamentous phages that can selectively infect F-pili bearing (F+) Escherichia coli (E.coli) cells. The phage genome encodes 11 proteins, while the phage coat itself consists of 5 of these proteins: gene3, -6, -7, -8 and -9 (g3, g6, g7, g8 and g9) proteins that are bound to and that protect the (circular) single stranded DNA (ssDNA) of the viral genome. The life cycle of the virus can be subdivided into different phases.
The g3 protein (g3p) of M13 phages and M13-derivatives comprises three functional domains: D1, D2 and D3, linked by two glycine-rich linkers. An alternative nomenclature for g3p domains has also been generally accepted, in which D1, D2 and D3 are named “N1,” “N2” and “CT,” respectively. The N-terminal D1/D2 regions interact with the C-terminal D3 region as has been found by Chatellier et al. (1999) using several deletion mutants of g3p. Considering that functionality of a D3 domain of the protein is required for assembly of stable phages, a less-, or non-infectious mutant of the phage coat protein preferably comprises a D3 region of the g3p, or comprises a functional part, derivative and/or analogue of the D3 region. The D3 domain is thought to bind to DNA inside the viral particle. Loss of the D3 domain functionally results in rare phage-like particles that are very long and very fragile (Pratt et al. 1969; Crissman and Smith. 1984; Rakonjac and Model. 1998). The D1 and D2 domain are thought to interact with each other until the phage binds to the bacteria, while D1/D2 also interact with D3 at certain stages (Chatellier et al. 1999). The linkers present in g3p between D1, D2 and D3 apparently also play a role in infectivity of the phage particle (Nilsson et al. 2000).
Studies in which a protease cleavage site was introduced between D1 and D2 showed that after cleavage, the phage particle became non-infectious (Kristensen and Winter. 1998). Functional analysis of g3p showed that of the g3p N-terminal regions, the D1 domain is essential for infection. Loss of this domain results in phages that cannot infect bacteria (Lubkowski et al. 1998; Nelson et al. 1981; Deng et al. 1999; Riechmann and Holliger. 1997; Holliger and Riechmann. 1997). It has been shown that the D2 domain interacts with the D1 domain of g3p on the phage (FIG. 1). Due to competition of proteins located on the F-pilus (on F+ bacteria) that have higher affinity for D2 than for D1, the D1 and D2 domain of the g3p dissociate from each other.
The binding of D2 to the F-pilus results in a process that leads to retraction of the F-pilus towards the E.coli cell membrane. Due to this process, the phage particle comes in close contact with the bacterial membrane. The dissociated D1 domain can interact with bacterial proteins such as the TolA receptor, leading to the introduction of the phage DNA into the E. coli cell (Lubkowski et al. 1999). The fact that removal of the D2 domain does not prevent infection, but enables phages to infect E.coli lacking F-pili (Riechmann and Holliger. 1997; Deng et al. 1999) shows that the presence of the D2 domain increases specificity and that D2 has an important role in preventing F-pili independent infections. The binding of D1 to the specific receptors on the surface of the E.coli cell (a feature that is not F+-specific) is represented in FIG. 2. This process triggers the injection of the viral genome into the bacterium (as depicted in FIG. 3).
Although loss of the D2 domain results in the formation of phage particles that can infect E.coli in a somewhat reduced specific manner, it appears that the level of infections from such a population of phages is significantly reduced. After infection of an E.coli by a phage particle, the ssDNA of the virus becomes double stranded due to the action of a number of bacterial enzymes. The double stranded phage genome serves as a template for the transcription and translation of all 11 genes located on the phage genome. Besides these protein-encoding regions, the phage genome contains an intergenic region: the F1-origin of replication initiation (F1-ORI). The DNA sequence of this F1-ORI can be divided in 2 separate subregions. One subregion is responsible for the initiation and termination of the synthesis of ssDNA via the so-called ‘rolling circle mechanism’ and the other subregion is responsible for the packaging initiation of the formed circular ssDNA leading to the formation and release of new virus particles.
It has been shown that polypeptides, such as stretches of amino acids, protein parts or even entire proteins can be added by means of molecular genetics to the terminal ends of a number of particle coat proteins, without disturbing the functionality of these proteins in the phage life cycle (Smith. 1985; Cwirla et al. 1990; Devlin et al. 1990; Bass et al. 1990; Felici et al. 1993; Luzzago et al. 1993).
This feature enables investigators to display peptides or proteins on phages, resulting in the generation of peptide- or protein expression phage display libraries. One of the proteins that has been used to fuse with polypeptides for phage display purposes, is the g3 protein (g3p), which is a coat protein that is required for an efficient and effective infectivity and subsequent entry of the viral genome into the E. coli cell.
For the production of phages that display polypeptides fused to the g3p coat protein, investigators introduced a plasmid together with the phage genome in E.coli cells. This plasmid contains an active promoter upstream of an in-frame fusion between the g3 encoding gene and a gene of interest (X) encoding, for instance, polypeptides such as proteins such as antibodies or fragments such as Fab fragments or scFv's. The introduction of this plasmid together with the genome of the helper phage in an E.coli cell results in the generation of phages that contain on their coat either the wild type g3p from the viral genome, the fusion product g3p-X from the plasmid or a mixture of the two, since one phage particle carries five g3p's on its surface. The process of g3p or g3p-X incorporation is generally random.
The presence of an F1-ORI sequence in the g3p-X expression vector (plasmid) misleads the phage synthesis machinery in such a way that two types of circular ssDNA are formed: one is derived from the genome of the phage and the other is derived from the expression vector. During the synthesis of new phages, the machinery is unable to distinguish the difference between these two forms of ssDNA resulting in the synthesis of a mixed population of phages, one part containing the phage genome and one part harboring the vector DNA. Due to these processes, the mixture contains at least some phages in which the phenotypic information on the outside (the g3p-X fusion protein) is conserved within the genotypic information inside the particle (the g3p-X expression vector). An infectious wild type phage and a phage carrying a fusion protein attached to g3p are depicted in FIG. 4. The art teaches that there are several problems that concern the use of these basic set-ups.
The high level of genotypic wild type phages in phage populations grown in bacteria that contain both the phage genome and the expression vector compelled investigators to design mutant F1-ORI sequences in M13 genomes. Such mutant M13-strains are less effective in incorporating their genome in phage particles during phage assembly, resulting in an increased percentage of phages containing vector sequences when co-expressed. These mutant phages, such as the commercially available strains R408, VCSM13 and M13KO7, are called “helper phages.” The genome of these helper phages may contain genes required to assemble new (helper-) phages in E.coli and to subsequently infect new F-pili expressing E.coli. Both VCSM13 and M13K07 were provided with an origin of replication initiation (ORI) of the P15A type that results in the multiplication of the viral genome in E.coli. Moreover, the ORI introduction ensures that after cell division the old and newly formed E.coli contains at least one copy of the viral genome.
It was suggested and finally proven by several investigators that the introduction of plasmids containing a g3p-scFv fusion product together with the genome of the helper phages in E.coli cells results in approximately 99% of newly formed phages that harbor the g3p-scFv fusion protein expression plasmid, but nevertheless lack the g3p-scFv fusion on its surface (Beekwilder et al. 1999). The absence of g3p-X is a significant disadvantage in the use of display libraries for the identification of specific proteins or peptides such as scFv's that bind to a target of interest (such as tumor antigens). It implies that in the case of phage display libraries, at least a 100-fold excess of produced phages must be used in an experiment in order to perform a selection with all possible fusion proteins present.
The art teaches that this overload of relatively useless phages in an experiment leads to (too) many false positives. For instance, at least 1012 phages should be added to a panning experiment in order to have 1 copy of each possible fusion present in the experiment, since such a library contains approximately 1010 different g3p-scFv fusions (1%). The phages in this approximate 1% express generally only 1 g3p-scFv fusion on their coat together with four normal g3p's (no fusions), while the rest of the helper phages (approximately 99%) express five g3p's and no g3p-scFv fusions. To ensure, theoretically, the presence of 100 copies of each separate fusion protein in a panning experiment, one needs to use approximately 1014 phages in such an experiment. Persons skilled in the art generally attempt to use an excess of at least 100-fold of each single unique fusion protein, to ensure the presence of sufficient numbers of each separate fusion and not to lose relevant binders too quickly in first panning rounds. That number of phages (1014) is more or less the maximum of phage particles that a milliliter (ml) can hold. The viscosity of such a solution is extremely high and therefore relatively useless. Especially when ELISA panning strategies are used (in which the volume of one well is only 200 μl) such libraries cannot be used.
In addition to these problems, it is assumed that, generally, depending on the antigen and the stringency of washing procedures, an average of 1 in every 107 phages will bind to the antigen due to a-specific binding. Thus, for the application of 1012 scFv expressing input phages (1%) to a panning procedure, one has to add approximately 1014 phages (99% of which do not express a scFv fragment). It is generally assumed that from these 1012 phages, approximately 104 particles might be putatively interesting phages. However, depending on the washing conditions, the number of calculated background phages that are normally found by using libraries present in the art after one round of panning, was approximately 106-107 while only a few of these phages appear to be relevant binders. This is one of the most significant problems recognized in the art: too many background phages show up as initial binders in the phage mix after the first round of panning, while only a few significant and interesting binders are present in this mix. Thus, the absolute number of isolated phages after one round of panning is clearly too high (106-107). Moreover, in subsequent rounds of panning, non-specific background phages remain present. In libraries used in the art, most of these non-specific binders will amplify on bacteria that, upon amplification, continue to in a second round of panning. Therefore, the art teaches that the background level of a-specifically binding phages and the total number of phages per ml in these types of libraries is unacceptably high and remains high during subsequent rounds of panning.
A possibility that was suggested by investigators in the art as a solution to the problem of obtaining too many background phages that lack a g3p-X fusion was to remove the g3p-encoding gene entirely from the helper phage genome. In principle, this system ensures that during phage synthesis in an E.coli cell (that received the g3-less phage genome and a g3p-X fusion protein expression vector), only g3p-X proteins are incorporated in the newly formed phage coat. By doing so, each synthesized phage will express five copies of the g3p-X fusion product and hardly any phages are synthesized that express the g3p alone or that express less than five g3p-X fusions. R408-d3 and M13MDΔD3 are two examples of g3-minus helper phages (Dueñas and Borrebaeck. 1995; Rakonjac et al. 1997). Because the genome of these phages is not capable of supporting g3p synthesis, phage particles that carry less than five g3p-X fusion proteins can hardly be formed; or, if formed, are found to be non-infectious due to instability, since the art teaches that five g3p's are necessary to ensure a stable phage particle.
To produce helper phages that do not contain the g3 gene, but that are nevertheless infectious and that can be used to generate libraries of phages that carry five g3p-X fusion proteins, and that lack phages with less than five g3p-X fusions, it was recognized in the art that an external source for g3p was required. Such a source can be a vector without F1-ORI but that nevertheless contains an active promoter upstream of the full open reading frame (ORF) of g3. One major problem that is recognized by persons skilled in the art is that after the generation step of producing newly formed helper phages lacking a g3 gene, the yield is dramatically low. In fact, the yield of all described systems is below 1010 phages per liter, meaning that for a library of 1010 individual clones, at least 100 liters of helper phage culture are necessary (NB: the helper phages need to be purified) in order to grow the library once. The art, thus, teaches that phage libraries generated with such low titers of helper phages are not useful for phage display purposes and that these libraries cannot be used for panning experiments. One method for complementation of g3p deletion phages was presented recently, in which wild-type g3p was provided by a nucleic acid encoding the wild-type g3p, wherein the nucleic acid was stably incorporated into the host cell genome (Rondot et al. 2001).
Phages that express deleted g3p's fused to heterologous proteins have been generated. For the construction of most conventional Fab libraries and some scFv phage display libraries, the D1 domain and parts of the D2 domain were removed to ensure a shorter fusion protein, which was considered in the art as a product that could be translated easier than a full length g3p linked to a full length Fab fragment. The shorter g3p part would not prevent the generation of a viable and useful helper phage. Of course, such phages still depend on full length g3p's that are present on their surface next to the deleted g3p fusion with the Fab fragment for functional infectivity of E.coli cells. Also, phages that express deleted g3p's fused to ligand-binding proteins have been generated that depend on their infectious abilities on antigens that were fused to the parts of g3p that were missing from the non-infectious phage particle (Krebber et al. 1997; Spada et al. 1997). These particles depend for their infectivity on an interaction between the ligand-binding protein, such as an antibody or a fragment thereof and their respective ligand (or antigen). However, this interaction-dependency reduces the efficiency of infection, due to elimination of a direct linkage between the g3p domains, and a general inhibitory effect of the soluble N-terminal part of g3p coupled to the antigen.
The g3-minus helper phages R408-d3 and M13MDΔD3 mentioned above lack a bacterial ORI and a selection marker in their genome. The absence of a selection marker in the g3-minus genomes has a significant effect on the production scale of helper phages, because it results in an overgrowth of bacteria that do not contain the helper phage genome. It is known that bacteria grow slower when infected with the helper phage or virus. Therefore, bacteria that lack the phage genome quickly overgrow the other bacteria that do contain the genome. Another effect of the lack of an ORI or a selection marker is that g3-minus phage genomes cannot be kept in dividing bacteria during the production and expansion of phage display libraries. This is a very important negative feature because overgrowth of bacteria that lost the phage genome or that never received one, appear to have a growth advantage over bacteria that do contain the phage genome. In addition, such ‘empty’ bacteria are not capable of producing any phage and as a result, the phage display vectors including fusion protein fragments in such helper phages lacking-bacteria are lost permanently.
As mentioned, the g3p's are thought to be essential for the assembly of stable M13-like phages and because of their crucial role in infection, g3p's should be provided otherwise when g3-minus helper phages are to be generated. There is a prejudice in the art against making phage display libraries that lack g3p's because phages lacking g3p's are not stable. Rakonjac et al. (1997) constructed a VCSM13 g3-minus helper phage in parallel to a R408 g3-minus helper phage and used helper plasmids with either the psp or the lac promoter upstream of a full length g3 sequence to substitute g3 during helper phage synthesis (Model et al. 1997). However, the art teaches that the lac promoter has the disadvantage that it cannot be shut off completely, even not in the presence of high concentrations of glucose (3-5%) in the medium (Rakonjac and Model. 1998).
An additional problem that is well known in the art is that even very low levels of g3p in E.coli can block infection of M13-like phages. Moreover, it has been shown that co-encapsidation of plasmids together with the phage genome can occur (Russel and Model. 1989; Krebber et al. 1995; Rakonjac et al. 1997). If co-encapsidation occurs with the lac driven helper plasmid, it will compete with the lac driven vectors used in the phage display resulting in the efficient production of infectious phage particles that will not contain the g3p-X fusion product. Together, the art thus teaches that the lac promoter is not the best candidate promoter in the helper plasmid system. The psp promoter has the advantage to be relatively silent in E.coli until infection (Rakonjac et al. 1997). Upon M13-class phage infection, the psp promoter becomes activated and the helper plasmid will produce g3 proteins. However, the disadvantage of this promoter is that the level of RNA production cannot be regulated with external factors, but has to be regulated by either mutating (and change the activity of) the promotor, changing the ribosomal binding site (RBS) or other elements that influence the promotor activity. To figure out the ideal level of promotor activity in a specific E.coli strain can be time consuming and needs to be optimized for each E.coli strain separately. The art also teaches that the psp promotor system is not very attractive for large-scale helper phage production due to the inflexibility of E.coli strains, the time consuming optimization and the significant low level of helper phage production.
A significant problematic feature helper phage systems described is the occurrence of unwanted recombination events between the helper genome and the (helper-) plasmids. The problem that confronts investigators in the art is the fact that the g3 DNA sequences in the helper phages are homologous to the g3 sequences in the phage display vector and/or the helper phage plasmid. This results, in many cases, in recombination between the two DNA strains and therefore loss of functionality of the library as a whole.